Prothymosin alpha is a small, highly acidic, abundant, protein which is essential for cell growth. It is found in the nuclei of all mammalian cells. Previous studies have shown that human prothymosin alpha molecules contain substoichiometric amounts of phosphoserine localized primarily on serine 1, that the protein acquires identical amounts of phosphate at all stages of the cell cycle, and that prothymosin alpha is metabolically stable [Sburlati et al. (1993) Biochemistry 32, 4587-96]. The half-life of the phosphates on prothymosin alpha has been measured in vivo with HeLa cells pulse labeled with [32P]orthophosphate and chased using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. In rapidly growing HeLa cells, the phosphate turned over with a half-life of approximately 90 min regardless of the method. The ability of cells to phosphorylate "old" prothymosin alpha molecules was confirmed by equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half- life of the phosphates is metabolically regulated, with phosphoprothymosin alpha in growing NIH 3T3 cells exhibiting twice the stability of that in quiescent cells. Further, these activities appear to be linked to synthesis of RNA because the ability of prothymosin alpha to acquire phosphate depends on active transcriptional elongation; in contrast, loss of phosphate is unaffected by inhibitors of transcription such as actinomycin D or alpha-amanitin. The properties of these phosphates are aberrant when compared with those of phosphotyrosine, phosphothreonine, or phosphoserine; the experimental results are consistent with a model in which the the phosphate acquired by prothymosin alpha transfers to serine 1. The data make possible the characterization of prothymosin alpha as a protein donor of phosphates and our experiments leave open the possibility that these unusual phosphates are transferred to a spectrum of acceptors in vivo.